Isoflavanoids comprise a large class of compounds, many of which have significant biological effects. Certain isoflavanoids belong to a broader class of phytoestrogens, compounds found in the plant kingdom that possess chemical structure and biological activity similar to estrogens. (see Merck Index, XII Edition, No. 4395).
Estrogens and phytoestrogens have overlapping, but not coextensive, effects on different tissues in the body. For example, estrogens have a much more pronounced effect on breast and uterine tissue than phytoestrogens, but both estrogens and phytoestrogens have strong salutory effects on blood vessels and bone. Although the mechanisms are not yet completely elucidated, the divergence has recently been explained as a function of the differing activities of these compounds upon different classes of estrogen receptors. Estrogens are reported to have a pronounced effect on both estrogen receptor alpha (ER.alpha.) predominantly expressed in breast and uterine tissue, and on estrogen receptor beta (ER.beta.) which is predominantly expressed in endothelial cells of blood vessels, and osteoclasts and osteoblasts of bone. In contrast, phytoestrogens have only a relatively small effect on ER.alpha., but a relatively great effect on ER.beta.. (Kuiper, G. G. M. J., Enmark, E., Pelto-Huikko, M., Nilsson, S., and Gustafsson, J-A., "Cloning of a novel estrogen receptor expressed in rat prostate and ovary", Proc. Natl. Acad. Sci. USA 93, 5925-5930, 1996; Mosselman, S., Polman, J., and Dijkema, R., "ER.beta.:Identification and characterization of a novel human estrogen receptor", FEBS Lett. 392, 49-53, 1996; Jan-Ake Gustafsson: "Estrogen receptor beta--Getting in on the action", Nature Medicine, 3 (number 5), 493-494 (May 1997), and lafrati, M. D., Karas, R. H., Aronovitz, M., Kim S., Sullivan, Jr., T. R., Lubahn, D. B., O'Donnell, T. F., Korach, K. S., and Mendelsohn M. E., "Estrogen inhibits the vascular injury response in estrogen receptor alpha-deficient mice", Nature Medicine, 3, (number 5), 545-548 (1997)).
Taken together, these findings explain many of the seemingly contradictory biological activities of estrogens and phytoestrogens. For example, since breast and ovarian tissue contains mostly ER.alpha. receptors, estrogens (as in estrogen replacement therapy) increases the risk of breast and ovarian cancer, while phytoestrogens do not. (Kuiper, G. G., Carlsson B., Grandien, K., Enmark, E., Haggblad, J., Nilsson, S., and Gustafsson J-A. "Comparison of the ligand binding specificity and transcript tissue distribution of estrogen receptors alpha and beta", Endocrinology, 138(3), 863-870 (1997)). Similarly, since bone deposition is controlled in part by ER.beta., both estrogens and phytoestrogens can increase bone density. (see, for example, Migliaccio, S., Davis V. L., Gibson, M. K., Gray, T. K., and Korach, K. S., "Estrogens modulate the responsiveness of osteoblast-like cells (ROS 17/2.8) stably transfected with estrogen receptor", Endocrinology, 130(5), 2617-2624 (1992); and Davis, V. L., Couse, J. F., Gray, T. K., and Korach, K. S., "Correlation between low level of estrogen receptors and estrogen responsiveness in two rat osteoblast-like cell lines", J Bone Miner Res 9(7), 983-991 (1994)).
In view of these findings, long-term nutritional supplementation with isoflavanoids, and in particular with phytoestrogens, has great potential. Moreover, while such supplementation has an especially important effect in women, the fact that ER.beta. is also present in male endothelial cells, osteoblasts and osteoclasts, leads to the conclusion that such compounds can also be useful in protecting men as well against arteriosclerosis and osteoporosis.
Among the more active phytoestrogens are genistin and genistein (see FIG. 1). These compounds are found in low concentration in soybeans, red clover, and several other plants, and ingestion of products made from these sources has indeed been associated with reduced incidence of circulatory and skeletal disease. Genistein and some other accompanying natural isoflavanoids (such as biochanin A) have also demonstrated chemopreventive activity in a variety of cancers (see, for example: Messina et al., Nutrition and Cancer, 21, 113-131, 1994; and Barnes, S., Sfakianos, J., Coward, L., and Kirk, M., in Dietary Phytochemicals in Cancer Prevention and Treatment, American Institute for Cancer Research, Plenum Press, New York, 1996, p87-100). Genistein and soybean isoflavanoids are especially effective in inhibition of cancer cell growth in breast (Pagliacci et all., Eur J Cancer 30A, 1675-1682, 1994) prostate (Peterson and Barnes, Prostate, 22, 335-345, 1993) and colon (Kuo, Cancer Letters, 110, 41-48, 1996). In addition, some genistein derivatives show pronounced curative properties in some type of cancer, apparently through growth inhibition of cancer cells (see, for example, the publication of Uckun et al, Science, 267, 886-891, 1995), describing genistein immunoconjugates that are highly efficient in treating B-cell precursor (BCP)-leukemia, a common form of childhood cancer). The mechanisms by which genistein and other related isoflavanoid compounds exhibit anti-cancer activity are believed to include tyrosine kinase(s) activity, topoisomerase II inhibition, antioxidant activity (free radical scavenging), angiogenesis inhibition, apoptosis induction and cell differentiation induction (see review by Peterson, J Nutr 125, 784S-789S, 1995).
Industrial mixtures of crude isoflavanoid are currently produced either as a side product of soybean processing, or by extraction of selected medicinal plants (like red clover). Contemporary nutritional supplement industry practice usually starts with such crude isoflavanoid mixtures, and then merely powders and compresses the mixtures into tablet and capsule forms. Alternatively, isoflavanoids are sometimes concentrated along with the protein components of soybeans into the well-known soybean based foods and drinks such as tofu, miso, and so forth.
The isoflavanoid content in such products is almost always low, typically ranging from between 0.02% and 1%. Only rarely is the isoflavanoid content in nutritional supplements increased, and then only to about 40% A great majority of these higher quality products are based on genistin or other isotlavanoid glucosides. Pure isoflavanoid is extremely expensive.
In addition to relatively low concentration of isoflavanoids, nutritional supplements and soybean based foods typically contain isoflavanoids in a form that has very poor bioavailability. The situation is exacerbated by the fact that genistein and its 7-O-glucoside, genistin, are readily biotransformed in hepato-biliary circulation to 7O-glucuronide and/or to the corresponding 7O-sulfate, which is excreted through the urine. As a result, isoflavanoid blood levels in humans are in sub-micromolar region, even with high intake of isoflavanoids. (see, for example, Barnes, S. et al. cited above).
Without being limited by the validity of any particular theory or practice, it is contemplated that genistein or analogous isoflavanoids must usually reach a blood concentration of between 1-10 micromoles/liter to achieve a desirable biological activity (such as maintaining the bones healthy, with a satisfactory bone density, keeping the blood vessels free from cholesterol plaques, or for the purpose of cancer chemoprevention, etc.) (Barnes, S. et al., cited above). Due to various combinations of low concentration and poor bioavailability of the active isoflavanoids in the supplements, the desired blood concentrations cannot realistically be achieved over the short term by administering any of the existing market preparations. Thus, there is a strong current need for an isoflavanoid preparation having improved concentration and bioavailability, and particularly for preparations providing improved concentration and bioavailability of genistein, genistein, and/or derivatives thereof.
Recently, the pharmaceutical industry introduced microemulsification technology to improve bioavailability of some lipophilic (water insoluble) pharmaceutical agents. Examples include Trimetrine (Dordunoo, S. K., et al., Drug Development and Industrial Pharmacy, 17(12), 1685-1713, 1991 and REV 5901 (Sheen, P. C., et al., J Pharm Sci 80(7), 712-714, 1991). Among other things, microemulsification provides enhanced bioavailability by preferentially directing absorption to the lymphatic system instead of the circulatory system, which thereby bypasses the liver, and prevents destruction of the compounds in the hepatobiliary circulation.
Interestingly, it appears that microemulsion formulations have never been used in the field of nutritional supplements. The likely reasons are that (1) microemulsion formulations are rather expensive relative to the price that can be charged for the end product, and (2) the number of GRAS (Generally Recognized As Safe) emulsifiers/co-emulsifiers and solvents/co-solvents that can be employed to implement microemulsification in the nutritional field is considerably smaller the corresponding number in the pharmaceutical field. An additional difficulty in the field of isoflavanoids is that relatively expensive concentration and purification steps are generally required to achieve proper microemulsification.
In short, there is a strong, ongoing need to provide relatively concentrated, and highly bioavailable formulations of isoflavanoids, especially of genistein, genistin, and derivatives thereof: